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HER-2/neu Expression in Primary Breast Cancer With Sentinel Lymph Node Metastasis

¹ Department of Surgery, Henry Ford Health System, 2799 West Grand Boulevard, Detroit, Michigan 48202
² Josephine Ford Cancer Center Clinical Trials Office, Henry Ford Health System, Detroit, Michigan 48202
³ Department of Biostatistics and Research Epidemiology, Henry Ford Health System, Detroit, Michigan 48202
⁴ Department of Pathology, Henry Ford Health System, Detroit, Michigan 48202

Correspondence: Address correspondence and reprint requests to: S. David Nathanson, MD; E-mail: dnathan1{at}hfhs.org.

	ABSTRACT

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Background: Amplification of the protein product of the HER-2/neu oncogene in primary breast cancer specimens is associated with an adverse prognosis. We hypothesized that overexpression of HER-2/neu would predict metastases to the sentinel lymph nodes (SLNs).

Methods: A retrospective review of a prospective nonrandomized evaluation of 1055 clinically node-negative breast cancer patients undergoing 1063 SLN biopsies was performed. HER-2/neu analysis was performed by immunohistochemistry and, in selected cases, by fluorescence in situ hybridization. Clinical, demographic, surgical, radiological, and pathologic data were analyzed by using generalized estimating equations logistic regression models.

Results: Two hundred thirty-two (23.6%) of 985 operations in which the SLN was found at operation resulted in positive nodes. In a multivariate analysis, size (P < .0001) and HER-2/neu overexpression (P = .026) were independent predictors of SLN metastasis.

Conclusions: Size is a known predictor of SLN metastasis in the modern SLN era, as it was in the pre-SLN eras. HER-2/neu was found to be significantly predictive of SLN metastasis in our study. We anticipate a future when even the relatively minor procedure of SLN biopsy might be avoided with the predictive information gained from studying the pathology and molecular markers of primary breast cancers.

Key Words: Sentinel lymph nodes • Breast cancer • HER-2/neu • Prediction

	INTRODUCTION

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Sentinel lymph node biopsy (SLNB), a common surgical procedure,^1–5 is an accurate⁶ and safe technique for staging breast cancer. Metastasis to the sentinel lymph node (SLN) is one of the most important prognostic factors for this disease⁷ and provides vital information for staging. Complete axillary lymph node dissection (ALND), the standard for approximately 100 years, provides pathologists a considerable number of lymph nodes for pathologic analysis, which can lead to a lower diagnostic yield than a small number of nodes would yield.⁸

Clinical and pathologic predictors of axillary lymph node metastasis have been evaluated in the past.^9–13 Surgeons performed ALND on almost all patients with invasive breast cancers, and the predictors were of some interest but did not alter the intention of performing a complete ALND. However, the advent of SLNB has helped us change our paradigm of thinking; we now anticipate a future in which even this relatively minor procedure might be avoided.^14,15 In the molecular age, it is not unreasonable to predict that evaluation of gene dysregulation¹⁶ in primary tumors might identify patients whose likelihood of SLN metastasis is so low that SLNB could be safely avoided.

Many of the current methods for detecting gene deletions, upregulations, or mutations are not used clinically in most pathology laboratories. However, it has become quite routine to examine and report the HER-2/neu expression in breast cancer because relatively simple and reproducible techniques are available to assess this oncogene; it has been shown to be of prognostic value,¹⁷ and it is predictive of responses to systemic therapy.^18,19 In the first major clinical study, multiple node involvement was significantly associated with HER-2/neu amplification in primary tumors.¹⁷ Some later studies looked at HER-2/neu overexpression in breast cancer patients and did not show a significant association with axillary lymph node metastasis.^9–13 These studies included multiple institutions, and the techniques and reporting of HER-2/neu expression may not have been uniform.

Vascular endothelial growth factor C (VEGF-C) is a key peptide that is overexpressed in breast cancer with lymphatic metastasis.²⁰ HER-2/neu overexpression correlates with increased expression of VEGF-C.²¹ Measurement of VEGF-C is currently not performed in most clinical laboratories. We hypothesized that HER-2/neu, because of its association with VEGF-C and the mechanisms involved in lymphatic metastasis, could be a predictor of SLN metastasis. We set out to explore this possibility in a prospective nonrandomized analysis of a large number of breast cancer patients undergoing SLNB.

	METHODS

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Patients
The diagnosis of breast cancer was made by core needle biopsy in the clinic by a surgeon (for palpable masses), by a radiologist in the mammography suite by using image-guided techniques (radiography or ultrasonography), or by excisional biopsy in the operating room by a surgeon. All cases were discussed at a breast cancer tumor board meeting by surgeons, medical oncologists, radiation oncologists, radiologists, and pathologists. Patients were seen in a multidisciplinary breast cancer clinic, and treatment options, including SLNB, were presented and discussed. The study presented in this article includes a prospective nonrandomized evaluation of 1055 clinically node-negative patients who underwent 1063 SLNBs from April 1995 up to and including May 2005. They include 131 patients in the American College of Surgeons Oncology Group (ACOSOG) sentinel node study Z0010.

SLNB for breast cancer at our institution was first performed in April 1995. The first 60 cases were performed during a learning phase, when an additional complete ALND was performed at the time of the SLNB. The procedure was performed with the approval of the institutional review board. The SLN was identified during surgery in 39 patients. The difficulty in identifying the other 21 cases was ascribable to the technique of ^99mTc-labeled sulfur colloid injection. In these initial cases, radiocolloid was injected into the breast parenchyma adjacent to the primary breast tumor and did not always drain to the SLN on gamma camera image. No radioactive node was identified during surgery. In the next 100 patients, the sentinel node was identified in 90 patients after the injection technique was changed to the intradermal site for the radiocolloid. One hundred forty more patients deliberately had both SLNB and ALND at the same operative time. Of the first 269 patients, 7 (2.6%) in whom the sentinel node was negative had a metastasis in at least 1 of the other axillary lymph nodes (unpublished data). In 1999, we changed policy and performed SLNB without a planned ALND on almost every new breast cancer patient who consented to have the procedure. Patients with a positive SLN were advised to have an ALND or to enroll in the ACOSOG Z0011 study, which randomized patients to either no further surgery or ALND. The 1055 patients in this study include all these patients up to May 2005.

The Technique of SLNB
Twenty-two board-certified surgeons were trained in-house to perform SLNB. Four surgeons continued to perform the procedure on a regular basis, 6 more performed at least 20 cases, and the remaining 12 surgeons performed 1 or 2 cases each. The quality of the SLNB was monitored during surgery for each of the first 10 cases by one of the authors (S.D.N.) for surgeons who continued to use the technique. Four surgeons, who performed most of the operations, were accredited by the ACOSOG after satisfactorily performing 30 SLNBs and ALNDs with no more than 1 false-negative SLN and a find rate >90%. Initially all SLNBs were performed at the main academic medical center, where lymphoscintigraphy was performed, as was isosulfan blue dye injection. The four surgeons accredited by the ACOSOG operated at suburban satellite surgical centers and also a downtown academic center. Lymphoscintigraphy was not available at the suburban satellite surgical centers, and blue dye alone was used to identify the SLN.

All operations were performed after appropriate consent was obtained by the operating surgeon. Many patients were seen at the weekly multidisciplinary breast cancer clinic by a surgeon, radiation oncologist, and medical oncologist. The consent process included a detailed discussion about the experimental nature of SLNB. A small number of our breast cancer patients elected to undergo ALND without SLNB, and they were excluded from this study.

The technique we used for SLNB was described previously.²² Briefly, a lymphoscintigram was obtained after intradermal injection of ^99mTc-labeled filtered sulfur colloid directly overlying the area of the tumor (except for the first 60 patients, as described previously). The radiologist marked the area of the SLN on the skin of the axilla after identifying the drainage pathway and the lymph node with a gamma camera. Four cases had drainage to the axilla and to the internal mammary nodes, but the latter were not removed during surgery. General endotracheal anesthesia was used in all cases. Intraoperative lymphatic mapping was performed after injection of 5 mL of 1% isosulfan blue into the breast parenchyma adjacent to the breast cancer. If the tumor in the breast had previously been removed, care was taken to avoid injection into the lumpectomy cavity. As our technique matured, we began to inject 1 mL into the subareolar space and 4 mL into the deeper breast parenchyma. The breast was massaged for 4 to 6 minutes. Attention was directed to opening the axilla to find a blue lymphatic and trace it to a blue lymph node. Only when these criteria for identification of the SLN had been satisfied did we use a gamma probe to confirm that the blue node was also radioactive (hot). In 6% (n = 22) of cases, the blue and the hot nodes were two different nodes, usually adjacent to each other. Additional blue and/or hot nodes were removed, occasionally yielding more than one SLN. A complete (level I and II) ALND was performed in the first 300 patients as part of the learning phase before we changed to a new era of offering SLNB alone to most of our patients. Most patients with a positive SLN underwent a subsequent ALND, although 12 patients elected to forego this procedure despite our recommendations. The primary breast tumor was excised by mastectomy or lumpectomy/ partial mastectomy/quadrantectomy by using standard techniques.

Pathologic Evaluation
All primary tumors and axillary lymph nodes were subjected to expert pathologic evaluation by a breast pathologist. Tumor size was measured both in the fresh state and after fixation and was recorded. Tumors and lymph nodes were fixed in 10% buffered formalin and paraffin-embedded. Five-micrometer slices were placed on glass slides, and standard hematoxylin and eosin staining was performed. The SLN was cut in half along the equator of the node, and four to six 5-µm slices were cut at various depths and stained on glass slides. Cytokeratin immunohistochemistry (IHC) was deliberately avoided except in cases in which a few suspicious cells were identified in the node and the pathologist wished to confirm that these were tumor cells. An axillary node was classified as positive according to current American Joint Commission on Cancer guidelines. Hormone receptor assays (both estrogen [ER] and progesterone receptors) were performed in a standard way by using IHC and were scored as either negative or positive.

HER-2/neu evaluations were performed with IHC and, selectively, by fluorescence in situ hybridization (FISH). The method used for measuring overexpression by IHC was according to the manufacturer’s instructions (Dako, Carpinteria, CA). The DAKO IHC system qualitatively identifies by light microscopy the P185 HER-2/neu overexpression in cancer cells by specifically binding to antigens located in the membrane of cells that overexpress the HER-2/neu gene product. The Food and Drug Administration–approved clinical laboratory assay was performed on deparaffinized, formalin-fixed tissue sections. Antibody to HER-2/neu protein (DAKO clone A0485; polyclonal antibody) was used with a DAKO automated immunostainer by following the manufacturer’s protocol (DAKO HercepTest code K 5204). Immunostained slides were reviewed by light microscopy, and the staining intensity pattern and percentage of immunoreactive cells were semiquantitatively assessed and scored according to the DAKO guidelines. Only the membrane staining intensity and pattern were evaluated and scored as follows: 0, no observed staining or some staining in <10% of tumor cells (negative); 1+, faint or barely perceptible membrane staining in >10% of tumor cells; only parts of the membrane were stained (negative); 2+, weak to moderate complete membrane staining in >10% of tumor cells (weakly positive); or 3+, strong complete membrane staining was observed in >10% of tumor cells (strongly positive). All assays were performed by the same pathologist.

All tumors graded as 1+ or 2+ were subjected to FISH. All tumors positive by FISH were classified as HER-2/neu positive. All tumors identified by IHC or FISH as negative were classified as negative for this study. HER-2/neu assays were not performed on the first 279 patients in this study, and no attempt was made to retrieve the tissue blocks on those patients in whom HER-2/neu had not been performed.

FISH was performed by using the PathVysion HER-2 DNA FISH kit (Vysis Inc., Downers Grove, IL) according to the manufacturer’s instructions. Two probes are incorporated in the kit: an orange probe directed against the HER-2 gene on chromosome 17 and a green probe against the pericentomeric region of chromosome 17. Acid and protease pretreatments were performed (Vysis paraffin pretreatment kit) followed by standard saline citrate and formamide denaturation (72°C for 5 minutes). After dehydration, the HER-2/CEP 17 probe cocktail was added, and coverslips were applied and sealed with rubber cement. The slides were covered in light-proof foil and then incubated in a humid chamber for 18 hours at 37°C. The slides were washed in a stringency buffer (standard saline citrate; NP-40) and allowed to dry in a dark area. The slides were then incubated with 4,6-diamidino-2-phenylindole for nuclear identification. Nonamplified and amplified control slides (PathVysion kit; Vysis Inc.) were analyzed with each assay. Slides were scored with a Zeiss Axioplan Epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY) equipped with a 100-W mercury arc lamp, by using a multifilter cube supplied by Vysis. Sixty nonoverlapping invasive cancer nuclei for both CEP 17 (green) and HER-2 (orange) signals were counted. Only cancer nuclei that showed at least one orange and one green signal were counted. A case was considered amplified if the HER-2:CEP17 ratio was 2.0. The FISH assays were read by one experienced pathologist, and photo print documentation was obtained.

Data Retrieval, Collection, and Abstraction
Demographic, clinical, operative, radiological, and pathologic information was carefully abstracted by the staff in the Clinical Trials Office in consultation with the principal investigator (S.D.N.) and recorded in an Excel spreadsheet (MicrosoftCorp., Redmond, WA). Regular meetings were conducted to discuss data input. The database was periodically updated and searched for errors, which were corrected. The database was used for analysis by a biostatistician (A.K.) in consultation with the principal investigator.

Statistical Methods
For all statistical analyses, generalized estimating equations (GEE) logistic regression models were used.^23,24 The GEE models were run by using PROC GENMOD in SAS version 9.0 (SAS Institute, Cary, NC). The GEE method takes into account the correlation in outcomes for patients seen by the same doctor. Doctors who performed fewer than 20 operations were grouped together, and this resulted in 11 doctor clusters. Eight patients had procedures on both sides. Analyses were run that included all 1063 procedures performed and were then run by randomly excluding 1 procedure among these 8 patients. Results were not different, and, therefore, all procedures were included in the final analyses.

We first tested each predictor of interest in a univariate GEE model. Individual predictors with P < .10 were candidates for multivariable modeling. The backward model selection method was used to determine the final multivariable model. Odds ratios (ORs) and 95% confidence intervals were calculated for the univariate models and the multivariable model. An OR of 1.0 indicates no association between the predictor and a positive SLN. An OR >1 indicates a positive association between a positive SLN and the predictor variable, whereas an OR <1 indicates a negative relationship between a positive SLN and the predictor.

	RESULTS

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Table 1 shows clinical, pathologic, and surgical data. The mean age was 63.7 years, with an SD of 12.9 years and a range of 32 to 94 years. There were 1055 patients, who underwent 1063 SLNBs. Five hundred nineteen patients had right-sided breast cancers, 527 had left-sided cancers, and 8 patients had bilateral breast cancers and had sentinel node biopsies on both sides. There were 2 men and 1053 women in this cohort. A total of 1043 cancers were invasive ductal or lobular cancers, and the other 10 were large (>4-cm) ductal carcinomas-in-situ. HER-2/neu assays were not performed in patients who underwent SLNB for ductal carcinomas-in-situ, and these patients were thus automatically excluded from the multivariable analysis for HER-2/neu.

Two hundred sixty-seven patients had total mastectomies with SLNB, and 796 had lumpectomies (also called partial mastectomy by some surgeons). Four hundred seventy-two patients had ALNDs, including 220 with positive sentinel nodes. Another 12 patients with positive sentinel nodes declined ALND. We did not record the complications for SLNB in our database, although we did have two memorable patients with an anaphylactic reaction to the blue dye that necessitated treatment.

Table 2 shows GEE results from the univariate tests for the variables HER-2/neu, ER, progesterone receptor, tumor size, tumor grade, and age in relation to SLN metastases. Other variables looked at and found not significant included the type of operation, whether dye alone or dye plus radio-colloid injection was used to find the node, and the surgeon performing the procedure. Results indicated that patients whose tumors were HER-2/neu positive had approximately a 50% higher chance of having SLN metastases than those whose tumors did not overexpress this marker. Also, tumor size was a significant individual predictor of SLN metastases. Compared with tumors 1 cm, those between 1 and 2 cm were 3.7-fold more likely to metastasize to the SLN. The tumors 2 to 5 cm were 7.3-fold more likely, and those 5 cm were 5.5-fold more likely to metastasize. The only other significant individual predictor of SLN metastases was tumor grade. Compared with grade 1 tumors, the odds of SLN metastases were 2.2 for grade 2 tumors and 2.4 for grade 3 tumors.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

SLNB has become a common procedure in the treatment of T1 and T2 clinically node-negative breast cancer to stage the axilla in academic cancer centers.^1–4 It is not used as commonly to manage breast cancer in community hospitals, low-use regions, or rural communities.⁵ SLNB has a sensitivity and specificity of >90%.⁶

Classic prognostic factors for breast cancer are those demographic, clinical, pathologic, biological, and molecular characteristics that are associated with tumor recurrence and survival.⁷ Axillary lymph node status is vital for the staging of breast cancer and is an important prognostic factor. The introduction of SLNB as a diagnostic procedure has improved the diagnostic accuracy of the axillary lymph node status.⁸ Most of the studies evaluating the likelihood of regional lymph node metastasis were performed when ALND was the standard of care. Our study looked afresh at those same variables in SLNB and also for possible new predictors of SLN metastasis. Because SLNB provides the pathologist a single node or a relatively small number of nodes, the likelihood of identifying positive nodes has increased.⁸ This could potentially alter the variables associated with SLN metastasis. Factors that predict SLN metastasis are essential to ascertain which patients are likely to have negative SLNs and, therefore, are unlikely to benefit from SLNB.

Much of the information in the literature on the clinical value of HER-2/neu in primary breast cancers has focused on its value as a prognostic variable for survival¹⁷ or as a predictor of response to the humanized monoclonal anti–HER-2/neu antibody trastuzumab.^18,19 Reported predictors of axillary lymph node status identified include tumor size, grade, lymphovascular invasion, and age,^9–13 but no significant association between HER-2/neu expression and positive SLNs had been identified.^13,25,26 One study did show that HER-2/neu overexpression was associated with multiple positive nodes.¹⁷ The discrepancy with our data may be a result of the initial lack of standardization of the assay methods, the lack of uniformity when HER-2/neu assays are performed in different laboratories, the relatively small number of patients in other studies, and the accuracy of lymph node analyses in studies that rely on ALND and its lower chance of identifying positive lymph nodes compared with SLNB. The advantage of our study is that only two people read the IHC assays and one person performed the FISH assays; the protocols for the assays were rigidly followed, and our overall positivity rate (14%) was relatively low compared with the reports in the literature.¹⁷ This suggests that a higher rate of breast cancer patients with HER-2/neu positive tumors may have included some that were falsely positive, especially if a confirming FISH assay was not performed. Such false-positive results could adversely skew the data analyzed.

HER-2/neu (c-erbB-2) is a member of the epidermal growth factor receptor (EGFR) family. The gene has been localized to chromosome 17q and encodes a transmembrane tyrosine kinase receptor protein.^27,28 HER-2/neu is overexpressed in 20% to 30% of invasive breast cancers.¹⁷ The HER-2/neu receptor protein is overexpressed in 50% to 60% of cases of ductal carcinoma in situ and in essentially 100% of large-cell, comedo-type tumors.²⁶ Multiple studies have been performed that show that HER-2/neu overexpression is associated with worse survival and a reduced overall time to relapse.^17,29,30 In patients whose tumors expressed HER-2/neu and who had positive lymph nodes, prognosis was significantly worse compared with those who were HER-2/neu negative and lymph node positive.³⁰ These findings have led to the conclusion that HER-2/neu overexpression is associated with a more aggressive subtype of cancer.

The mechanisms whereby HER-2/neu overexpression in breast cancer results in a worse prognosis are not clear. Biochemical and molecular studies have been hampered by the lack of a ligand for the receptor. The role HER-2/neu plays in the development and progression of tumors has not been clearly identified. Because HER-2/neu amplification is associated with a poor prognosis in breast cancer patients, it is intuitive that it probably plays a role in the steps leading to hematogenous metastasis. Many of the early steps in tumor invasion into blood vessels are the same as those that are required for lymphatic metastasis.²⁰

Genes that encode receptor tyrosine kinases of the EGFR family, including EGFR, HER-2/neu, ERBB3, and ERBB4, are commonly dysregulated in cancer.³¹ Activation of HER-2/neu is associated with EGFR expression. Although EGFR has not been directly linked to lymphatic metastasis, other receptor tyrosine kinases have. VEGF-C and fibroblast growth factor induce lymphangiogenesis, a vital component of metastasis to regional lymph nodes.²⁰ Some studies have linked HER-2/neu overexpression with increased expression of VEGF.^21,32 In one study, VEGF121-206 was overexpressed in 77.2% of patients with HER-2/neu overexpression versus 54.5% in patients without overexpression of HER-2/neu.³² Similarly, overexpression of HER-2/neu was associated with overexpression of VEGF165-206 in 87.7% of patients, compared to 71.0% who did not overexpress HER-2/neu. Overexpression of VEGF, either alone or in combination with HER-2/neu, is associated with decreased relapse-free survival and overall survival.^33,34 Upregulation of HER-2/neu may be causally associated with upregulation of VEGF and the mechanisms of metastasis,²⁰ or the genes for these receptors may be mutated independently as part of the biology of tumor induction.

The mechanisms involved in SLN metastasis are complex and interrelated.²⁰ VEGF-C is produced by tumor cells and some host cells. Paracrine secretion of this peptide induces lymphangiogenesis, which is associated with an increase in regional lymph node metastasis. An interdependent HER-2/neu and VEGF-C relationship has been reported in human breast cancers, thus suggesting that these may be causally related to SLN metastasis. No direct association between HER-2/neu and lymphangiogenesis or any other factors involved in the complex steps of lymphatic metastasis has been described, so the actual pathogenesis awaits future discovery.

ER-negative tumors overexpress members of the epidermal growth factor family, especially EGFR and HER-2/neu. When HER-2/neu is activated in experimental systems, ER expression is suppressed.³⁵ In our study, although there was an inverse relationship between ER and HER-2/neu expression, ER expression alone did not predict which patients would have positive SLNs, thus confirming the findings of others investigating this variable at a time when SLNB was not performed routinely.^10,11

Primary tumor size was a significant predictor of SLN metastases in our study. This variable has been previously identified as an important prognostic and predictive variable in breast cancer,⁷ and it continues to be an important predictor of SLN metastasis.

Our study suffers from the common pitfalls seen with a retrospective analysis, although it maintains the strength of prospective data collection. We had a 2.6% false-negative SLN rate in our first 300 cases; we do not know whether the rate is the same in our last 763 cases because the patients with negative SLNBs did not undergo a second operation to remove the remaining axillary lymph nodes. HER-2/neu data rely on the accuracy of the assay, which is dependent on multiple laboratory factors and on the expertise of the pathologists reading the IHC and FISH slides. Ongoing data collection and improved methodologies will enable us to reassess the role this oncogene plays in prediction of SLN metastases.

The simplifications of breast cancer surgical management over the past few decades¹⁴ help us anticipate a future when not only ALND but also SLNB may be avoided. Simple pathologic information from needle biopsies of primary tumors may be enough to determine which patients may not significantly benefit from SLNB.¹⁵ Our HER-2/neu data are not compelling enough to incorporate the information into patient management decisions at this time. Perhaps future molecular information, such as the multigene array technology recently reported,¹⁶ plus the addition of HER-2/neu data, shown here to be a significant predictor of SLN metastases, will even further enhance our ability to safely omit SLNB in the future.

	ACKNOWLEDGMENTS

 
Supported by the Nathanson/Rands Endowment for Breast Cancer Research.

Received for publication March 4, 2005. Accepted for publication August 23, 2005.

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